Normal-subtracted preprocessing of Raman spectra aiming to discriminate skin actinic keratosis and neoplasias from benign lesions and normal skin tissues

The differences in the biochemistry of normal and cancerous tissue could be better exploited by Raman spectroscopy when the spectral information from normal tissue is subtracted from the abnormal tissues. In this study, we evaluated the use of the normal-subtracted spectra to evidence the biochemical differences in the pre-cancerous and cancerous skin tissues compared with normal skin, and to discriminate the groups with altered tissues with respect to the normal sites. Raman spectra from skin tissues [normal (Normal), benign (dermatitis-BEN), basal cell carcinoma (BCC), squamous cell carcinoma (SCC), and actinic keratosis (KER)] were obtained in vivo (Silveira et al., 2015, doi: 10.1002/lsm.22318) and used to develop the spectral model. The mean spectrum of the normal sites (circumjacent to each lesion) from each subject was calculated and subtracted from each individual spectrum of that particular subject independently of the group (Normal, BEN, BCC, SCC, KERAT). The mean spectra of each altered group and the mean spectra of the differences were firstly evaluated in terms of biochemical contribution or differentiation comparing the normal site. Then, the normal-subtracted spectra were submitted to discriminant models based on partial least squares and principal components regression (PLS-DA and PCR-DA), and the discrimination were compared with the model using non-subtracted spectra. Results showed that the peaks of nucleic acids, lipids (triolein) and proteins (elastin and collagens I, III, and IV) were significantly different in the lesions, higher for the pre- and neoplastic lesions compared with normal and benign. The PLS-DA showed that the groups could be discriminated with 90.3% accuracy when the mean-subtracted spectra were used, contrasting with 75.1% accuracy when the non-subtracted spectra were used. Also, when discriminating non-neoplastic tissue (Normal + BEN) from pre- and neoplastic sites (BCC + SCC + KERAT), the accuracy increases to 92.5% for the normal-subtracted compared with 85.3% for the non-subtracted. The subtraction of the mean normal spectrum from the subject obtained circumjacent to each lesion could significantly increase the diagnostic capability of the Raman-based discrimination algorithm.